Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells.
Proteins involved in chromatin-interacting processes, like gene transcription, DNA replication, and DNA repair, bind directly or indirectly to DNA, leading to their immobilisation. However, to reach their target sites in the DNA the proteins have to somehow move through the nucleus. Fluorescence recovery after photobleaching (FRAP) has been shown to be a strong approach to study exactly these properties, i.e. mobility and (transient) immobilisation of the proteins under investigation. Here, we provide and discuss detailed protocols for some of the FRAP procedures that we have used to study protein behaviour in living cell nuclei. In addition, we provide examples of their application in the investigation of the androgen receptor (AR), a hormone-inducible transcription factor, and of two DNA-maintenance factors, the telomere binding proteins TRF1 and TRF2. We also provide protocols for qualitative FRAP analysis and a general scheme for computer modelling of the presented FRAP procedures that can be used to quantitatively analyse experimental FRAP curves.
van Royen
Martin E
ME
Department of Pathology, Josephine Nefkens Institute, Erasmus University Medical Centre, Rotterdam, The Netherlands.
Farla
Pascal
P
Mattern
Karin A
KA
Geverts
Bart
B
Trapman
Jan
J
Houtsmuller
Adriaan B
AB
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*
D009687:Nuclear Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins